Phenol Extraction

    Perhaps the most basic of all procedures in molecular cloning is the purification of nucleic acid . The key step, the removal of proteins, can often be carried out simply by extracting aqueous solutions of nucleic acids with phenol. Such extractions are used whenever it is necessary to inactivate or remove enzymes (e.g., restriction endonucleases) that are used in one step of an operation before proceeding to the next.

The standard way to remove proteins from nucleic acid solution is to extract once with phenol and once with chloroform (Sevac). This procedure takes advantage of the fact that deproteinization  is more efficient when two different organic solvents are used instead of one. Furthermore, although phenol denatures proteins efficiently, it does not completely inhibit RNase activity[81]. The final extraction with chloroform also removes any lingering traces of phenol from the nucleic acid preparation.

The sample is diluted to a volume of about 400 to 500 l. One volume TE-saturated phenol is added, mixed by vortexing. A centrifugation step is performed to separate layers (room temperature, 2min, 13000 rpm). The upper aqueous layer is removed and transferred to a new tube. The lower organic layer and interface is discarded. The aqueous layer is mixed with 0.5 volume Sevac  (chloroform:isoamyl alcohol 24:1) and centrifuged for 2 minutes at 13000 rpm. The aqueous layer is removed and collected in a new tube[82, 83].